), 102 U/ml (), 103 U/ml (•), 104 U/ml (), 105 U/ml (♦), and 106 U/ml (+). (more ...)
To study the morphological alterations that may occur during trypanolysis, TNF-α–treated parasites were analyzed by TEM and flow cytometry (FSC analysis). Concordant with the lysis experiments, TNF-α–induced morphological alterations occurred suddenly and progressed very fast (Fig. 2). Indeed, after 1 to 3 h of incubation with TNF-α, TEM analysis of the parasites revealed no major morphological changes (Fig. 2, Aa and Ab) and only a minor shift in FSC signals (~5%) was recorded by flow cytometric analysis after 3 h of incubation (Fig. 2 Cb). At this time point, light microscopy analysis only indicated that a minor population became nonmotile. After 4 h, an important part of the trypanosome population analyzed in TEM manifested gross morphological alterations in the presence of TNF-α. As shown in Fig. 2 Ac, normal cells were observed in the vicinity of cells with swollen organelles and ruptured plasma membranes, reminiscent of an osmotic shock treatment. Concomitantly, the FSC signal of a major part of the parasites shifted to the left (Fig. 2 Cc) and indicated a trypanolysis of ~40%. After 5 h of incubation, TEM analysis showed a far progressed lysis, although some completely intact cells were still found to be present (Fig. 2 Ad). FSC analysis indicated a trypanolysis in the range of 85% (Fig. 2 Cd). Control parasites that were incubated in the absence of TNF-α and analyzed in TEM showed no changes in morphology during the course of this experiment (Fig. 2 B, a–d). During trypanolysis experiments, we never recorded a TNF-α specific lysis >85%, not even when incubations were performed for periods up to 8 h. This observation indicates that within an apparently morphologically homogenous population of monomorphic trypanosomes, there is a heterogeneity with regard to TNF-α sensitivity.
| Figure 2Morphological analysis by TEM and FSC of TNF-α–induced trypanolysis in function of time (a, 1 h, b, 3 h, c, 4 h, d, 5 h). Purified trypanosomes were incubated for various periods of time in PSG (pH 8.0) in the presence or absence (more ...) |
Quantitative Analysis of TNF-α Binding on Live Trypanosomes and Trypanosome Lysate
To perform a quantitative analysis of the binding of TNF-α to live monomorphic AnTat 1.1 trypanosomes, parasites were incubated for 6 h at 4°C with different molar concentrations of 125I-TNF-α. The results shown in Fig. 3 a confirm a specific binding of 125I-TNF-α on bloodstream form trypanosomes. Competition binding experiments with 125ITNF-α in the presence of a 100-fold molar excess of cold TNF-α showed a significant reduction of the parasite labeling. No specific TNF-α binding could be recorded on procyclic parasites. Analysis of the specific binding data obtained with bloodstream form trypanosomes (Fig. 3 b) revealed an affinity constant of 37.2 ± 14.8 nM and 1,558 ± 302 TNF-α–specific binding sites. To confirm the binding of TNF-α to trypanosomes, a different method was adopted. Using an optical biosensor, the direct binding of TNF-α on total trypanosome lysate was measured (Fig. 4). To test whether this binding reflects an interaction between the lectin-like activity of TNF-α (25) and glycosylated trypanosome components, the trypanosome lysate was treated with N-glycosidase F. This treatment strongly reduced the binding of TNF-α to trypanosome lysate, indicating that indeed a glycoprotein of bloodstream-form trypanosomes binds TNF-α with high specificity via its N-linked carbohydrate moiety. | Figure 3Binding of 125ITNF-α to bloodstream-form and procyclic trypanosomes. (a) Bloodstream-form trypanosomes () and procyclic trypanosomes (□) were incubated in the presence of different molar concentrations of 125I-TNF-α. (more ...) |
| Figure 4The binding between TNF-α and total trypanosome lysate (1) or N-glycosidase F–treated trypanosome lysate (2) was analyzed using a biosensor. Sample injection was done at t = 0, and lysate was allowed to bind to a TNF-α (more ...) |
Ultrastructural Analysis of TNF-α Binding and Uptake
To localize TNF-α binding sites on intact parasites and to follow intracellular uptake of TNF-α, the cytokine was conjugated to 10-nm colloidal gold particles and subsequently incubated with monomorphic AnTat 1.1 trypanosomes at 30°C. The bulk of the TNF-α–gold labeling was localized in the flagellar pocket, where beads concentrated in coated pits (Fig. 5 a, arrowhead). Sporadically, TNFα–gold particles were found in association with the flagellum in the flagellar adhesion zone (Fig. 5, b and c), at the entrance of the flagellar pocket, or in association with tiny filamentous material at more distant regions of the flagellum (Fig. 5 d). After binding, gold labeled TNF-α was endocytosed through coated vesicles (Fig. 5 e). Coated vesicles containing TNF-α–gold particles were often seen fusing with larger electrolucent vacuoles wherein a cytoplasmic area was invaginated, assuming cup-like shapes (Fig. 5 f) which might look like rings in TEM (Fig. 5 g). Gold particles were also observed in tubular vesicular structures (Fig. 5, h and i, top) but more frequently in dilatations of the collecting membrane system described by Langreth and Balber (21) which contain electron-dense material (Fig. 5 i, lower part, j and k, middle). In parasites incubated for longer periods (1 or 2 h), gold particles were also localized in larger lysosome-like digestive vacuoles with more (Fig. 5 k, top, and l) or less (Fig. 5 m) electron-lucent contents. These vacuoles often sequestered areas of cytoplasm remaining probably in connection with the surrounding ground cytoplasm. As shown in Fig. 5 n, the vacuole from a partially lysed parasite contains some gold particles (arrow) in addition to several such cytoplasmic areas. | Figure 5Localization by TEM of TNF-α binding and internalization at 30°C by T. brucei. TNF-α was conjugated to 10-nm gold particles as described in Materials and Methods. Cells were incubated with TNF-α–gold particles (more ...) |
When procyclic parasites were incubated with TNF-α–gold particles, no binding of TNF-α was seen in the flagellar pocket lumen, although rare formation of coated pits by the membrane of the flagellar pocket was observed (Fig. 5 o). Consequently, no internalization of gold particles was seen. As expected, no electron-dense inner layer, corresponding to the VSGs in the bloodstream forms (compare Fig. 5, o with e), was observed in procyclic coated pits.
When BSA–gold was used as a control, gold particles were only found in some intracellular vacuoles but not in coated pits or vacuoles. Competition experiments with an excess of unconjugated TNF-α, reduced substantially the cellular labeling, demonstrating that the binding of labeled gold particles was TNF-α specific. Similar inhibition of binding of TNF-α–gold particles was observed when the particles were preincubated with anti-TNF/TIP antibodies or N,N′diacetyl-chitobiose before incubation with the trypanosomes (data not shown). These data are concordant with our previous report that the lytic activity of TNF-α is mediated via the lectin-like TIP-domain (25).
Influence of Temperature and Intracellular pH on TNF-α–mediated Lysis
Incubations of parasites at 4°C with TNF-α–gold particles yielded a very rare labeling of the lumen of the flagellar pocket, and at this temperature no lysis was recorded, not even after 24 h of incubation (results not shown). To evaluate to which extent TNF-α–mediated trypanolysis is temperature dependent, experiments with monomorphic AnTat 1.1 trypanosomes were performed in parallel at different temperatures ranging from 37° to 17°C. According to the results shown in Fig. 6, the same maximal lysis was obtained at 37°, 32°, or 29°C, although the incubation time required to reach the lytic plateau value increased slightly with lower temperatures. At 26°C, ~50% of the maximal TNF-α–mediated lysis was recorded, and practically no lysis was observed at lower temperatures, not even when samples were incubated up to 18 h. | Figure 6TNF-α–mediated lysis of bloodstream forms of T. brucei as function of temperature and time. A lysis assay was performed using a TNF-α concentration of 104 U/ml. Samples were kept at the indicated temperatures for various periods (more ...) |
TNF-α–induced morphological alterations which preceded lysis could only be observed by light microscopy when incubation temperatures above 26°C were used. At 30°C in the absence of TNF-α, virtually no morphological changes were observed after a 5 h incubation period (Fig. 7 a). However, in the presence of TNF-α, a large number of cells were lysed, and most of the remaining cells showed a clearly altered swollen morphology (Fig. 7 b). When incubations were performed at 21°C, no morphological differences were observed between the cells incubated in the absence and presence of TNF-α, as shown in Fig. 7, c and d, respectively.
| Figure 7Morphological characteristics of temperature dependent TNF-α–mediated lysis of bloodstream forms of T. brucei. Lysis assays were performed for 5 h at 30° and 21°C as described in Materials and Methods, using a TNF-α (more ...) |
To analyze whether the insensitivity of the parasites to TNF-α–mediated lysis recorded at lower temperatures reflects inefficient TNF-α binding and/or uptake, monomorphic trypanosomes were incubated during 1 h at 17° or 21°C with TNF-α and subsequently washed and transferred to 30°C for another 4 h of incubation. In a reversed experimental setting, parasites were incubated with TNF-α for 1 h at 30°C and subsequently washed and transferred to 17° or 21°C. Control parasite populations were incubated with TNF-α during 1 h at 17°, 21°, or 30°C, washed, and further incubated at their initial temperatures. The results, shown in Table I, clearly indicate that during the first hour of incubation at 17° or 21°C, enough TNF-α was bound and internalized to yield a similar lysis as the lysis recorded during continuous incubation at 30°C. However, lysis was blocked at 17° and 21°C, even when the initial incubation was performed at 30°C.
| Table I TNF-mediated Trypanolysis in Function of Temperature |
Similar experiments were performed with TNF-α–gold particles, i.e., trypanosomes were incubated for 1 h with TNF-α–gold at 17° or 30°C. Subsequently, the samples were washed and reincubated during 4 h at the respective temperatures. TEM analysis shown in Fig. 8 indicates that parasites preincubated with TNF-α–gold particles during 1 h at 30°C, started lysing after a total incubation time of 4 h at this temperature. (Fig. 8 a). In contrast, parasites preincubated at 17°C with TNF-α–gold particles, and kept at this temperature for another 3 h, did not exhibit any sign of lysis, although TNF-α–gold complexes were clearly internalized (Fig. 8 b). At 30°C, lysis was found to be preceded by swelling of the vesicles containing TNF-α–gold particles and of the mitochondria. Often, vesicles containing TNF-α–gold particles exhibit large disruptions of their membranes (Fig. 8 a, arrow). Such events preceded prompt lysis. At 17°C, gold conjugates were found to be endocytosed, yet swelling of TNF-α–collecting organelles or lysis of cells was not recorded (Fig. 8 b).
| Figure 8Transmission electron microscopy analysis of intracellular uptake of TNF-α–gold particles and lysis of bloodstream forms of T. brucei, isolated at the early stage and the peak of the parasitaemia. Cells were incubated with TNF-α–gold (more ...) |
The TEM analysis with TNF-α–gold particles strongly suggests that the particles reach a lysosome-like compartment as a final destination. Hence, the influence of pH elevation on trypanolysis was analyzed. As shown in Fig. 9 a, the presence of NH4Cl during TNF-α incubations resulted clearly in a dose-dependent inhibition of lysis. Maximal inhibitions of ~90% were recorded using concentrations of 1 mM of NH4Cl or higher. Inhibition of TNF-α–mediated lysis by 1 mM of NH4Cl was even recorded when the compound was added 2 h after incubation with TNF-α (Fig. 9 b). Addition of NH4Cl after 3 h of TNF-α treatment did not result in a substantial inhibition of lysis. Hence, an acidic environment at the destination site of internalized TNF-α appears to be required for TNF-α–mediated lysis.
| Figure 9Inhibition of TNFα–mediated trypanolysis by NH4Cl. Lysis assays were carried out as described in Materials and Methods. (a) NH4Cl effects on lysis of T. brucei as function of concentration. Trypanosomes were incubated at 30°C (more ...) |
TNF-α–mediated Lysis of T. brucei Is Developmentally Regulated
To analyze whether different developmental stages of T. brucei differ in sensitivity towards TNF-α, lysis experiments were performed at 30°C on both monomorphic and pleomorphic AnTat 1.1 trypanosomes isolated at the initial phase of the parasitaemia and at the peak of the parasitaemia. As shown in Fig. 10, a and b, TNF-α–mediated lysis of both monomorphic and pleomorphic trypanosomes occurred only when these bloodstream parasites were isolated at the peak of the parasitaemia, while no lytic effect was observed when parasites were isolated at the beginning of the infection. These results point again to a heterogeneity of trypanosomes with respect to their susceptibility to TNF-α. | Figure 10Trypanolytic activity of TNF-α on both monomorphic and pleomorphic bloodstream forms of T. brucei. Lysis assays were carried out at 30°C in the presence of different concentrations of TNF-α as described in Materials and Methods. (more ...) |
Taking into account the possible role of lysosome damage as an early event in TNF-α–mediated trypanolysis, both early (Fig. 11, a and b) and late stage trypanosomes (Fig. 11, c and d) were stained with the fluorescent lysosome marker LysoTracker™ in the absence or presence of TNF-α. Both the morphology of the parasites and the intracellular localization of the marker were analyzed after 3 h of incubation at 30°C. First, Fig. 11 shows that lysed parasites (arrow) and parasites with altered morphology (arrowhead) were only observed when late stage parasites were treated with TNF-α (Fig. 11 Ad). No cell damage was observed in the other samples (Fig. 11 A, a–c). Furthermore, Fig. 11 Aa (early stage) and Ac (late stage) show that in the absence of TNF-α the lysosome marker was localized essentially in a single spot. In the presence of TNF-α, a similar localization was still observed in the early stage parasites (Fig. 11 Ab), while a diffuse staining was observed in the late stage parasites (Fig. 11 Ad). These results suggest that TNF-α–mediated damage of the trypanosomal lysosome-like organelles precedes total lysis of the cells and is developmentally regulated. To exclude the possibility that the lack of lysosomal damage in early stage trypanosomes was due to a lack of TNF-α uptake, these parasites were also incubated with TNF-α–gold particles for a total of 4 h at 30°C. Again, none of the events preceding trypanolysis were observed. As shown in Fig. 8 c, in such parasites, TNF-α–gold particles were endocytosed and subsequently collected in vesicles in a similar way as observed with peak stage parasites (Fig. 8 a), but no subsequent swelling or lysis of the cells was observed. These results stress again that the uptake of TNF-α is a necessary but not sufficient step to culminate in trypanolysis. Together with the earlier observed lack of TNF-α endocytosis by procyclic trypanosomes (Fig. 5 o) and the lack of TNF-α–mediated trypanolysis of these forms (data not shown), all results indicate that only late stage bloodstream-form trypanosomes are TNF-α susceptible while other forms are completely refractory to the trypanolytic activity of TNF-α.
| Figure 11TNF-α–mediated lysis of bloodstream forms of T. brucei is preceded by the destruction of their lysosome-like organelles. Both early and late stage trypanosomes were incubated for 3 h at 30°C with the lysosomal marker LysoTracker™ (more ...) |
Influence of Monoclonal Anti–TNF-α Antibodies on TNF-α–mediated Trypanolysis In Vitro
To confirm the involvement of the previously described lectin-like TNF/TIP domain in TNF-α–mediated trypanolysis (25), new monoclonal anti-TNF/TIP antibodies were elicited against TNF/TIP peptides encompassing the lectin-like activity of TNF-α. Antibodies were initially selected based on their affinity in ELISA for the TIP peptides used for immunization. For the selected monoclonal antibodies 1E12 and 24C11, the affinity for native TNF-α was further checked using the biosensor technique previously used to measure the affinity between trypanosome lysate and TNF-α. The dissociation constant values recorded were 32.7 ± 4.1 nM and 160.4 ± 67.9 nM for 1E12 and 24C11, respectively. As shown in Fig. 12, both antibodies and a previously described polyclonal rabbit anti-TIP antibody (25) were capable of neutralizing the trypanolytic effect of TNF-α in vitro (Fig. 12 a), while no inhibition activity was observed in a classical L929 TNF-α tumor lysis assay (Fig. 12 b). | Figure 12Specific inhibition of TNF-α–mediated lysis of bloodstream forms of T. brucei by anti-TIP antibodies. (a) Freshly isolated bloodstream-form trypanosomes were incubated for up to 8 h in PSG (pH 8.0) at 30°C in the presence (more ...) |
Influence of Anti–TNF-α Antibodies on T. brucei Development In Vivo
To evaluate the in vivo relevance of the in vitro TNF-α–mediated trypanolytic activity, T. brucei-infected mice were treated with the above described anti-TNF/TIP antibodies. We have reported earlier that the neutralizing monoclonal anti–TNF-α antibody 1F3F3 increases the number of parasites present in the blood of infected mice, indicating that TNF-α plays a controlling role during the normal course of a T. brucei parasitaemia (24). These experiments have now been repeated with the above described monoclonal and polyclonal anti-TNF/TIP antibodies. The results presented in Table II indicate that the in vivo treatment of T. brucei infected mice with the TIP-specific monoclonal antibodies 1E12 and 24C11 or the TIP-specific polyclonal antibody, resulted in a dramatic increase in the number of parasites during the first peak of the parasitaemia, as did the treatment with the 1F3F3 monoclonal anti–TNF-α antibody. The number of parasites, compared to controltreated animals, increased in the bloodstream, the spleen, the lymph nodes, and the peritoneal cavity. These results suggest that in vivo, TNF-α exerts a growth limiting effect on T. brucei via its lectin-like TIP domain and thus that the herein described trypanolytic activity of TNF-α is physiologically relevant. | Table II Influence of Anti-TNF Antibodies on Parasite Development in Trypanosoma-infected Mice |
Discussion The herein described data extends and corroborates our previous findings on the trypanolytic activity of the cytokine TNF-α on African trypanosomes (24–26). Furthermore, evidence is provided for a direct involvement of TNF-α in the growth regulation of T. brucei in its mammalian host. TNF-α induces the lysis of trypanosomes via a process that occurs suddenly and proceeds quickly after about 4 h of incubation at 30°C. This process of lysis is preceded by cellular swelling as if the cells are subjected to an osmotic shock. Hereby it should be emphasized that even when high concentrations of TNF-α (up to 106 U/ml) were used along with long incubation times (up to 8 h), a variable proportion of trypanosomes (15–30%) was found to be refractory towards TNF-α–mediated lysis. TNF-α lysis assays performed on monomorphic and pleomorphic AnTat 1.1 T. brucei parasites, isolated at the early parasitaemia phase or at the peak of the parasitaemia, revealed that the sensitivity of trypanosomes towards the lytic activity of TNF-α is marked by a destruction of the trypanosomal lysosome-like organelles and is developmentally regulated. As TNF-α sensitivity is only acquired at the peak of the parasitaemia, the minor population of nonsensitive trypanosomes still present during the peak, might represent a population that did not yet reach this sensitive stage. Since both monomorphic and pleomorphic trypanosomes, isolated at the peak of the parasitaemia, were found to be equally TNF-α sensitive, a differential TNF-α susceptibility of long slenders versus short stumps is excluded. Furthermore, procyclic trypanosomes were found to be completely resistant towards TNF-α. This lack of TNF-α sensitivity of the procyclic form is due to the lack of TNF-α binding. Scatchard analysis of 125I-TNF-α binding on bloodstream form trypanosomes revealed the presence of ~1,600 TNF-α–binding molecules, while no binding could be observed on procyclic parasites. Analyzing the binding between crude parasite lysates and TNF-α with an optical biosensor confirmed the presence of a bloodstream stage specific glycoprotein that is capable of binding TNF-α. Removal of N-linked carbohydrate groups by N-glucosidase F treatment strongly reduced the TNF-α binding. This observation confirms our previous finding that the lectin-like domain of TNF-α is involved in the interaction with trypanosomes (25). Using TNF-α–gold particles, binding of TNF-α was localized mainly in the flagellar pocket. This binding could be inhibited by cold TNF-α, anti-TNF/TIP antibodies, and N,N′diacetyl-chitobiose, indicating that the binding of gold particles is TNF-α specific and that the lectin-like domain of TNF-α is implicated in the binding process (25). Hence, TNF-α interacts most probably with specific carbohydrate components of the glycoprotein matrix enclosed in the flagellar pocket. In fact, WGA, which displays a similar carbohydrate specificity as TNF-α (35), was reported to bind selectively to the flagellar pocket of T. brucei (6), and we have shown that preincubation of T. brucei with WGA inhibits the trypanolytic activity of TNF-α (25). We found that TNF-α binds to the flagellar pocket and the flagellar adhesion zone of bloodstream forms but not of procyclic forms. Since the flagellar pocket glycoprotein composition was reported to differ between these two forms (13), it may be that TNF-α binding oligosaccarides are either absent or inaccessible in the flagellar membrane microdomains of procyclic trypanosomes. After binding, TNF-α–gold particles were found to be endocytosed through coated pits and vesicles. In bloodstream-form trypanosomes, this pathway is used for instance for the specific uptake of transferrin via its heterodimeric glycoprotein receptor (30). Coated vesicles containing TNF-α–gold were found to fuse with endosomes, and subsequently the particles were localized in tubular vesicular extensions and more electron-dense cisternae of the collecting membrane system, all structures that were extensively described in trypanosome bloodstream forms (21). Finally, TNF-α–gold complexes were found in digestive vacuoles connected to the collecting membrane system. Globally, the intracellular pathway of endocytosed TNF-α–gold complexes seems to involve similar structures as those ones associated with the specific uptake of transferrin (30, 37), ferritin (21), high density lipoprotein (HDL; 14), and low density lipoprotein (LDL; 7), suggesting that the final destination of the particles is lysosome-like digestive vacuoles. Disruption of these collecting vacuoles was frequently observed independent of whether native ligand (TNF-α) or ligand conjugates (TNF-α–gold) were tested in the trypanolysis assay. The exact mechanism underlying TNF-α–mediated trypanolysis is so far not defined. It is however clear that binding and endocytosis of TNF-α is necessary but not sufficient to induce trypanolysis. Indeed, endocytosis of TNF-α is comparable in early and peak stage bloodstream forms, yet only the peak-stage trypanosomes can be lysed by the cytokine. Furthermore, while TNF-α uptake occurs at 30° as well as at 21° and 17°C, its lytic activity appears to be strongly temperature dependent and requires a temperature >25°C. Hence an intracellular process that is developmentally regulated and temperature sensitive determines whether TNF-α–mediated lysis will or will not occur. Interestingly the threshold temperature required to inhibit the lytic activity of TNF-α does not correspond to that one preventing the fusion of endosomes and lysosomes (17°C) as documented for high density lipoprotein-mediated trypanolysis (14). Rather, this 25°C threshold temperature corresponds exactly to the temperature at which a shift in the membrane fluidity of trypanosomes occurs (20). Our results further indicate that TNF-α–mediated trypanolysis is pH dependent, since incubations with ammonium chloride strongly inhibit the lytic activity of TNF-α. Apparently an acid pH is required to allow TNF-α to exert its intracellular lytic activity. This observation may be related to the documented pore-forming capacity of TNF-α (18). Indeed at low pH, as a result of a conformational shift, TNF-α is able to integrate into mammalian membranes resulting in pore formation (3). Furthermore, the pore-forming capacity of TNF-α is mediated by the TIP-domain that is implicated in the binding of TNF-α on trypanosomes (18). The formation of ion-permeable channels could account for the influx of cytosolic ions into TNF-α–collecting organelles, leading to the features of osmotic shock that appear to be the primary event in TNF-α–mediated trypanolysis. After organelle rupture, release of proteolytic enzymes may accelerate the lytic process resulting in prompt and complete lysis. Hereby mitochondria could be among the first affected organelles, since circular cristae were frequently observed in mitochondria of TNF-α–treated cells. It should be emphasized that the proposed mechanism of TNF-α is not confined to trypanosomes but could also occur in mammalian cells. First, bypassing the classical TNF-α pathway involving the p55 and p75 TNF receptors by microinjection of the cytokine leads to lysis of mammalian cell lines such as L929 cells (36). Remarkably, intracellular administration of TNF-α causes lysis of L929 cells after 4 h (similar for lysis of trypanosomes), while when TNF-α is supplied in the medium, lysis of L929 cells requires ~20 h. Second, as already mentioned, TNF-α exerts a pH dependent pore-forming activity on mammalian membranes. Third, degeneration of mitochondrial structures is an early event in TNF-α–mediated lysis of mammalian cells (31). So intracellular TNF-α may exert similar activities in trypanosomes and mammalian cells. Till now, we cannot exclude the possibility that part of the intracellular TNF-α is recycled to the membrane and exocytosed. This possibility may account for our observation that clear TNF-α concentration dependent plateau levels of lysis were recorded, since partial recycling of intracellular TNF-α could result in a steady-state situation. The in vivo experiments with anti-TNF/TIP antibodies finally demonstrate that TNF-α exerts a growth-controlling function on trypanosomes. This host cytokine, which can be induced by the parasite itself (26, 38), apparently binds specifically on trypanosomes and kills certain developmental stages of the parasite. Though we cannot yet explain why TNF-α sensitivity is developmentally regulated, it is clear that trypanosomes are only lysed by TNF-α during the peak stage of the parasitaemia. Important to mention here is the recent report that TNF-α was shown to abolish the growth promoting effect of IFN-γ (2). Our findings show that in addition to counteracting growth promotion, TNF-α may contribute to limit the number of parasites in the bloodstream, peritoneal cavity, and lymphoid organs by actively lysing trypanosomes. |
Acknowledgments We thank Dr. Paul Voorheis, Dr. Derek Nolan, and Mrs. Magdalena Radwanska for their interest in this work and their helpful suggestions. We also thank Miss Lea Brys and Miss Martine Gobert for their technical assistance, as well as Mrs. Ella Omasta and Mr. Eddy Vercauteren for their secretarial assistance. |
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